Nucleic acid encoding n-methylputrescine oxidase and uses thereof

ABSTRACT

The gene encoding N-methylputrescine oxidase (MPO) and constructs comprising such DNA are provided, including methods of regulating MPO expression independently or with other alkaloid biosynthesis genes to modulate alkaloid production in plants and host cells. MPO genes or fragments thereof are useful for reducing pyrrolidine or tropane alkaloid production in plants, for increasing pyrrolidine or tropane alkaloid production in plants, and for producing an MPO enzyme in host cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/774,902, filed Feb. 22, 2013, now U.S. Pat. No. 9,422,532, which is adivisional of U.S. patent application Ser. No. 12/305,483, filed Feb.12, 2009, now U.S. Pat. No. 8,410,341, which is the National Phase ofInternational Patent Application No. PCT/IB2007/003550, filed Jun. 19,2007, which claims the benefit of priority from U.S. Provisional PatentApplications No. 60/814,542, filed Jun. 19, 2006, and 60/901,654, filedFeb. 16, 2007. The contents of these applications are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to molecular biology and alkaloidbiosynthesis in plants, plant cells or other cells. The inventionrelates, inter alia, to nucleic acid sequences that encodeN-methylputrescine oxidase (MPO) and to methods for their use inmodifying alkaloid production in plants, particularly but notexclusively nicotinic alkaloid production in a tobacco plant, and forproducing alkaloid biosynthetic enzymes in plant cells or other cells.

BACKGROUND OF THE INVENTION

Pyrrolidine alkaloids (e.g., nicotine) and tropane alkaloids (e.g.,scopolamine and cocaine) are plant natural products that exhibit adiverse range of pharmacological activities. For example, nicotine mayhave utility for increasing cognitive function and is used in nicotinereplacement therapy for smoking cessation. Tropane alkaloids areimportant anticholinergic drugs. Cocaine is used as a local anesthetic.These compounds are all isolated from plant sources for use aspharmaceutical drugs.

It would be of interest to enhance the production of pyrrolidine andtropane alkaloids in plants or plant cells by genetic engineering forthe more efficient commercial production of these compounds for thepharmaceutical industry. Enhanced production of pyrrolidine and tropanealkaloids could be accomplished through selective breeding or geneticengineering using genes that encode enzymes in the biosynthetic pathwaysleading to these alkaloids. Genetic engineering for the accumulation ofpathway intermediates, or reduction of end-product alkaloid levels,could be accomplished through classical or targeted mutagenesisapproaches, such as Targeting Induced Local Lesions in Genomes(TILLING), or the specific silencing of genes encoding these enzymes viaRNA interference and related techniques.

It would also be of interest to block the biosynthetic pathways leadingto the production of these alkaloids at defined metabolic steps. Thepurpose of this would be to accumulate metabolic intermediates that maybe of high value themselves or to generate plants that contain modifiedor reduced levels of end-product alkaloids. Tobacco plants that aregenetically engineered to contain reduced nicotine levels may be usefulfor the production of plant-made pharmaceuticals, reduced-nicotine ornicotine-free cigarettes for use as smoking cessation aids (Benowitz etal. Clin Pharmocol Ther. 80(6):703-14 (2006)) and for use aslow-toxicity industrial, food or biomass crops.

There are few genes known that encode enzymes involved in pyrrolidine ortropane alkaloid biosynthesis. This limits the ability to geneticallyengineer the pathways leading to these useful molecules.

An example of a known gene that encodes an enzyme involved inpyrrolidine alkaloid biosynthesis is the quinolate phosphoribosyltransferase (QPT) gene which has been cloned from N. tabacum and N.rustica; see U.S. Pat. No. 6,423,520 and U.S. Pat. No. 6,586,661, andSinclair et al., Plant Mol. Biol. 44: 603-17 (2000). QPT suppressionprovides significant nicotinic alkaloid reductions in transgenic tobaccoplants. Xie et al., Recent Advances in Tobacco Science 30: 17-37 (2004).

U.S. Pat. No. 5,369,023, and No. 5,260,205 discuss decreasing nicotinelevels by suppressing putrescine N-methyltransferase (PMT) sequence.Suppression of an endogenous putrescine N-methyltransferase (PMT)sequence has been shown to reduce nicotine levels and increase anatabinelevels by about 2-to-6-fold. Hibi et al., Plant Cell 6: 723-35 (1994);Sato et al. Proc Natl Acad Sci USA 98:367-72 (2001); Chintapakorn andHamill, Plant Mol. Biol. 53:87-105 (2003); Steppuhn of al., PLoS Biol.2:8:e217: 1074-1080 (2004). Overexpression of Nicotiana tabacum PMT inN. sylvestris resulted in an increase in nicotine content (Sato et al.Proc Natl Acad Sci USA 98:367-72 (2001)).

Suppression of endogenous A622 and NBB1 sequences has been shown toreduce nicotinic alkaloid levels in tobacco plants; see Internationalpatent publication WO 2006/109197. A gene encoding a cytochrome P450monooxygenase that converts nicotine to nornicotine has been cloned fromN. tabacum. Siminszky B., et al., Proc Natl Aced Sci USA 102:14919-24,(2005); Gavilano et al. J. Agric. Food Chem. 54, 9071-9078 (2006).

The enzymatic activity of MPO was first detected in tobacco roots overthree decades ago, Mizusaki S et al., Phytochemistry 11: 2757-2762(1972), but the gene encoding this enzyme has remained unidentifieduntil the present invention.

MPO plays a role in the pathway for the biosynthesis of alkaloids inplants, including medicinal tropane alkaloids. Hashimoto and Yamada,Annu. Rev. Plant Physiol. Plant Mol. Biol. 45: 257-285 (1994); Kutchan,In: Cordell, Ga. (ed.) ALKALOIDS (San Diego), vol. 50. Academic Press,Inc., San Diego, Calif., USA, (1998) pp. 257-316.

MPO enzymes have been purified from the roots of N. tabacum and N.nistica, Hyoscyamus niger, and Bnigmansia candidax aurea hybrid and wereshown to oxidize N-methylputrescine more efficiently than putrescine andcadaverine. Mizusaki et al., Phytochemistry 11: 2757-2762 (1972); Fethand Wagner, Phytochemistry 24: 1653-1655 (1985); Davies et al.,Phytochemistry 28: 1573-1578 (1989); Hashimoto et al. Plant Physiol.93:216-221 (1990); Walton and McLauchlan, Phytochemistry 29: 1455-1457(1990); Haslam and Young, Phytochemistry 3 1:4075-4079 (1992);McLauchlan et al., Planta 19: 440-445 (1993); Boswell et al.,Phytochemistry 52: 871-878 (1999).

Anabasine and anatalline contain a piperidine moiety. The piperidinemoiety of anabasine is thought to be derived from cadaverine viadelta-1-piperidine in tobacco. Watson and Brown, J. Chem. Soc. PerkinTrans. 1: 2607-2610 (1990). Cadaverine is a good substrate for generaldiamine oxidases but is also a substrate for MPO, although it has alower affinity than N-methylated diamines. Hashimoto et al. (1990),supra; Walton and McLauchlan, Phytochemistry 29: 1455-1457 (1990);Boswell et al., Phytochemistry 52: 871-878 (1999).

Katoh et al., Plant Cell Physiol. 48(3): 550-554 (2007), and Heim atal., Phytochemistry 68:454-463 (2007), both of which published after thefiling of U.S. provisional application Ser. No. 60/814,542, disclosegenes from tobacco that encode N-methylputrescine oxidase (MPO), whichis involved in the nicotine biosynthetic pathway. There is no teachingof any method or use involving this gene for modifying nicotineproduction in plants, or for modifying production of any other alkaloidin plants.

Accordingly, there is a continuing need to identify additional geneswhose expression can be regulated to decrease or increase thebiosynthesis of alkaloids or to alter a plant's alkaloid profile byregulating the biosynthesis of a specific alkaloid(s).

SUMMARY OF THE INVENTION

In one aspect, the invention provides an isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence set forth in SEQ ID NO: 1; (b) a nucleotidesequence set forth in SEQ ID NO: 2; (c) a nucleotide sequence thatencodes a polypeptide having the amino acid sequence set forth in SEQ IDNO: 3; (d) a nucleotide sequence that is at least 85% identical to thenucleotide sequences of (a), (b), or (c) and encodes an MPO enzyme; (e)a nucleotide sequence that hybridizes under stringent conditions to thenucleotide sequences of (a), (b), (c), or (d) and encodes an MPO enzyme;and (f) a nucleotide sequence that differs from the nucleic acidsequence of (a) or (b) due to the degeneracy of the genetic code andencodes an MPO enzyme.

In another aspect, the invention provides a method of producing MPOenzyme, comprising (a) genetically engineering a cell with a nucleicacid construct comprising the isolated nucleic acid molecule of claim 1;and (b) growing the engineered cell under conditions such that MPO isproduced.

In another aspect, the invention provides a recombinant MPO enzymehaving the amino acid sequence of SEQ ID NO: 3 or a variant of SEQ IDNO: 3.

In one embodiment, a genetically engineered host cell comprises thenucleic acid sequence. In a further embodiment, the host cell isselected from the group consisting of bacteria, yeast, filamentousfungi, algae, green plants, and mammalian cells. In another embodiment,a plant comprises the host cell.

In another aspect, the invention provides a method for reducing analkaloid in a plant, comprising down-regulating N-methylputrescineoxidase expression relative to a control plant. In one embodiment, thealkaloid is a pyrrolidine alkaloid. In a further embodiment, thepyrrolidine alkaloid is nicotine. In another embodiment, the plantbelongs to genus Nicotiana. In another embodiment, the plant isNicotiana tabacum. In another embodiment, N-methylputrescine oxidaseexpression is down-regulated by (a) introducing into the plant anucleotide sequence comprising i) at least 21 consecutive nucleotides ofSEQ ID NO: 1, wherein said consecutive nucleotides are in either senseor antisense orientation; and (b) growing the plant under conditionswhereby said nucleotide sequence decreases levels of N-methylputrescineoxidase in the plant compared to a control plant grown under similarconditions. In one embodiment, the conditions induce co-suppression ofan endogenous MPO gene.

In another aspect, the invention provides a method for reducing apyrrolidine alkaloid in a plant, comprising down-regulating MPO and atleast one of NBB1, A622, QPT and PMT.

In another aspect, the invention provides a method for decreasingpyrrolidine alkaloid levels in a population of plants, comprising (a)providing a population of mutated plants; (b) detecting a target mutatedplant within said population, wherein said target mutated plant hasdecreased expression of N-methylputrescine oxidase gene or decreasedactivity of N-methylputrescine oxidase enzyme compared to a controlplant provided under similar conditions, said detecting comprising usingprimers developed from SEQ ID NO: 1 or SEQ ID NO: 2 to PCR amplifyregions of the N-methylputrescine oxidase gene from mutated plants inthe population of mutated plants, identifying mismatches between theamplified regions and corresponding regions in wild-type gene that leadto the decreased expression of N-methylputrescine oxidase gene ordecreased activity of N-methylputrescine oxidase enzyme, and identifyingthe mutated plant that contains the mismatches; and (c) selectivelybreeding the target mutated plant to produce a population of plantshaving decreased expression of N-methylputrescine oxidase gene ordecreased activity of N-methylputrescine oxidase enzyme compared to apopulation of control plants produced under similar conditions.

In one embodiment, the pyrrolidine alkaloid is nicotine. In anotherembodiment, the plant is Nicotiana tabacum.

In another aspect, the invention provides a host cell geneticallyengineered with a nucleotide sequence comprising at least 21 consecutivenucleotides of SEQ ID NO: 1, wherein said consecutive nucleotides are ineither sense or antisense orientation. In one embodiment, the cell is acell of a plant from a member of family Solanaceae or familyEfythroxylaceae. In another embodiment, the cell is a cell of a plantfrom a member of genus Nicotiana, Datura, Atropa, Duboisia, Hyoscyamus,Mandragora, Brugmansia, Scopolia or Erythroxylon. In another embodiment,the cell is a cell of a plant from a member of the Nicotiana genus. Inanother embodiment, the cell is a cell of a plant of the speciesNicotiana tabacum. In a further embodiment, the nucleotide sequencecomprises SEQ ID NO: 1 or SEQ ID NO: 2.

In another embodiment, there is provided a reduced alkaloid plantproduced by any of the preceding methods. In another embodiment, thereduced alkaloid is nicotine. In a further embodiment, a reducedalkaloid product is produced from the reduced alkaloid plant. In anotherembodiment, the reduced alkaloid is nicotine.

In another aspect, the invention provides a method of increasing MPOenzyme in a plant comprising (a) introducing into the plant a nucleicacid construct comprising a nucleotide sequence encoding an MPO enzyme:and (b) growing the plant under conditions whereby the nucleotidesequence is expressed thereby increasing levels of MPO enzyme in theplant compared to a control plant grown under similar conditions.

In one embodiment, the plant is a member of family Solanaceae or familyErythroxylaceae. In another embodiment, the plant is a member of genusNicotiana, Datum, Atropa, Duboisia, Hyoscyamus, Mandragora, Brugmansia,Scopolia or Erythroxylon. In another embodiment, the level of a tropanealkaloid level is increased, and said tropane alkaloid comprises cocaineor scopolamine.

In another aspect, the invention provides a method of increasingpyrrolidine alkaloid levels in a plant, comprising (a) introducing intothe plant a nucleotide sequence encoding an MPO enzyme and a nucleotidesequence encoding at least one enzyme selected from the group consistingof NBB1, A622, QPT and PMT; and (b) growing the plant under conditionswhereby the plant produces increased levels of N-methylputrescineoxidase and at least one enzyme selected from the group consisting ofNBB1, A622, QPT and PMT compared to a control plant grown under similarconditions.

In one embodiment, the invention provides an increased alkaloid plantproduced by any of the preceding methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts proposed biosynthetic pathways leading to the pyrrolidinealkaloid, nicotine, and the tropane alkaloids, cocaine and scopolamine.

FIG. 2 depicts RT-PCR analysis of MPO expression in different tissues ofNicotiana benthamiana.

FIG. 3 depicts quantitative RT-PCR (qRT-PCR) analysis of PMT and MPOexpression in roots of N. benthamiana in response to treatment withmethyljasmonate (MeJa).

FIG. 4 depicts positions and lengths of MPO gene fragments used for VIGSsilencing of nicotine production in N. benthamiana plants relative tothe full-length MPO cDNA.

FIG. 5A depicts nicotine content of leaves sampled from control plantsand plants treated with MPO VIGS vectors as determined by HPLC analysis.

FIG. 5B depicts changes in nicotine levels in control plants and plantsinfected with TRV-MPO construct 403B01 in response to methyljasmonate(MeJa) application.

FIG. 6 depicts an HPLC analysis of N-methylputrescine in root tissue ofplants treated with an MPO VIGS vector or with a control vectorcontaining GFP sequences.

FIG. 7 depicts MPO transcript level in roots of plants treated with anMPO VIGS vector.

FIG. 8 depicts MPO activity in roots of plants treated with an MPO VIGSvector.

FIG. 9 depicts substrate specificity and enzyme kinetics of recombinantMPO.

FIG. 10 depicts mass spectrometry analysis of the MPO product.

FIG. 11 depicts GC-MS analysis of derivatized MPO product.

FIG. 12 depicts gel electrophoresis analysis of PCR amplicons fromplants transformed with an MPO overexpression construct.

FIG. 13 depicts leaf nicotine levels in wild-type and transgenic plantsoverexpressing MPO.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have cloned a gene encoding N-methylputrescineoxidase (MPO). The nucleic acid sequence of the MPO gene, SEQ ID NO: 1,has been determined. The open reading frame (ORF) of SEQ ID NO: 1, setforth in SEQ ID NO: 2, encodes the polypeptide sequence set forth in SEQID NO: 3.

With reference to FIG. 1, the biosynthesis of both pyrrolidine alkaloids(e.g., nicotine) and tropane alkaloids (e.g., cocaine and scopolamine)involves N-methylpyrrolinium on as a metabolic intermediate. It isapparent that the production of these alkaloids can be modulated byaffecting the enzymes and/or intermediates within these pathways.

MPO catalyzes the oxidative deamination of N-methylputrescine to form4-methylaminobutanal, which spontaneously cyclizes to yieldN-methylpyrrolinium ion. N-methylpyrrolinium ion is a key building blockfor valuable alkaloids. FIG. 1 shows the structure of the key metabolicintermediate, N-methylpyrrolinium ion, as well as its positions in thestructures of nicotine, cocaine, and scopolamine. The two known enzymes,quinolinate phosphoribosyl transferase (OPT) and putrescineN-methyltransferase (PMT), also are indicated.

Assuming there are sufficient levels of N-methylputrescine available, itis apparent from FIG. 1 that increasing MPO levels and/or activity willincrease N-methylpyrrolinium ion levels, which in turn will result inthe production of more nicotine, cocaine and/or scopolamine.Alternatively, nicotine, cocaine and/or scopolamine biosynthesis will bedecreased if MPO levels and/or activity are reduced sufficiently.

The MPO gene or fragments thereof may be used to suppress pyrrolidinealkaloid biosynthesis (e.g., of nicotine) in plants that naturallyproduce the pyrrolidine alkaloids. For example, Nicotiana spp. (e.g. N.tabacum, N. rustica and N. benthamiana) naturally produce nicotine. N.tabacum remains an agricultural crop of high value and biotechnologicaluses of this plant continue to increase. Blocking nicotine biosynthesisby MPO suppression leads to creating tobacco varieties that contain zeroor low nicotine levels for use as low-toxicity production platforms forthe production of plant-made pharmaceuticals (PMPs) (e.g. recombinantproteins and antibodies) or as industrial, food and biomass crops.

DEFINITIONS

All technical terms employed in this specification are commonly used inbiochemistry, molecular biology and agriculture; hence, they areunderstood by those skilled in the field to which this inventionbelongs. Those technical terms can be found, for example in: MOLECULARCLONING: A LABORATORY MANUAL 3rd ed., vol. 1-3, ed. Sambrook and Russel,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001;CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, ed. Ausubel et al., GreenePublishing Associates and Wiley-Interscience, New York, 1988 (includingperiodic updates); SHORT PROTOCOLS IN MOLECULAR BIOLOGY: A COMPENDIUM OFMETHODS FROM CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 5th ed., vol. 1-2,ed. Ausubel et al., John Wiley & Sons, Inc., 2002; GENOME ANALYSIS: ALABORATORY MANUAL, vol. 1-2, ed. Green et al., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1997. Methodology involvingplant biology techniques are described here and also are described indetail in treatises such as METHODS IN PLANT MOLECULAR BIOLOGY: ALABORATORY COURSE MANUAL, ed. Maliga et al., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1995.

The terms “encoding” and “coding” refer to the process by which a gene,through the mechanisms of transcription and translation, providesinformation to a cell from which a series of amino acids can beassembled into a specific amino acid sequence to produce an activeenzyme. Because of the degeneracy of the genetic code, certain basechanges in DNA sequence do not change the amino acid sequence of aprotein.

As used herein, “expression” denotes the production of an RNA productthrough transcription of a gene or the production of the protein productencoded by a nucleotide sequence.

Overexpression” or “up-regulation” is used to indicate that expressionof a particular gene sequence or variant thereof, in a cell or plant,including all progeny plants derived thereof, has been increased bygenetic engineering, relative to a control cell or plant (e.g., “MPOoverexpression”).

The terms “suppression” or “down-regulation” are used synonymously toindicate that expression of a particular gene sequence variant thereof,in a cell or plant, including all progeny plants derived thereof, hasbeen reduced by genetic engineering, relative to a control cell or plant(e.g., “MPO down-regulation”).

An “alkaloid” is a nitrogen-containing basic compound found in plantsand produced by secondary metabolism. A “pyrrolidine alkaloid” is analkaloid containing a pyrrolidine ring as part of its molecularstructure, for example, nicotine. Nicotine and related alkaloids arealso referred to as pyridine alkaloids in the published literature. A“pyridine alkaloid” is an alkaloid containing a pyridine ring as part ofits molecular, structure, for example, nicotine. A “tropane alkaloid” isan alkaloid containing a bicyclic tropane ring system as part of itsmolecular structure for example, scopolamine or cocaine. A “nicotinicalkaloid” is nicotine or an alkaloid that is structurally related tonicotine and that is synthesized from a compound produced in thenicotine biosynthesis pathway. Illustrative nicotinic alkaloids includebut are not limited to nicotine, nornicotine, anatabine, anabasine,anatalline, N-methylanatabine, N-methylanabasine, myosmine, anabaseine,formylnornicotine, nicotyrine, and cotinine. Other very minor nicotinicalkaloids in tobacco leaf are reported, for example, in Hecht, S. S. ofal., Accounts of Chemical Research 12: 92-98 (1979); Tso, T. C.,Production, Physiology and Biochemistry of Tobacco Plant. Ideals Inc.,Beltsville, Md. (1990).

As used herein “alkaloid content” means the total amount of alkaloidsfound in a plant, for example, in terms of pg/g dry weight (DVV) orng/mg fresh weight (FW). “Nicotine content” means the total amount ofnicotine found in a plant, for example, in terms of mg/g DW or FW.

“Decreased nicotine tobacco plant” or “reduced nicotine tobacco plant”encompasses a genetically engineered tobacco plant that has a decreasein nicotine content to a level less than 50%, and preferably less than10%, or 1% of the nicotine content of a control plant of the samespecies or variety.

“Increased nicotine tobacco plant” encompasses a genetically engineeredplant that has an increase in nicotine content greater than 10%, andpreferably greater than 50%, 100%, or 200% of the nicotine content of acontrol plant of the same species or variety.

“MPO activity” is the enzymatic oxidation of N-methylputrescine to form4-methylaminobutanal and hydrogen peroxide catalyzed by the enzymeN-methylputrescine oxidase.

An “N-methylputrescine oxidase,” “MPO” or “MPO enzyme” is an enzyme thatoxidases N-methylputrescine to form N-methylaminobutanal.

I. Reducing Alkaloid Production in Plants

A. Decreasing Alkaloids by Suppressing MPO.

Alkaloid (e.g. nicotine) production may be reduced by suppression ofendogenous MPO genes using the MPO sequence of the present invention ina number of ways generally known in the art, for example, RNAinterference (RNAi) techniques, artificial microRNA techniques,virus-induced gene silencing (VIGS) techniques, antisense techniques,sense co-suppression techniques and targeted mutagenesis techniques.Accordingly, the present invention provides methodology and constructsfor decreasing alkaloid content in a plant, by suppressing an MPO gene.Suppressing more than one MPO gene may further decrease alkaloids levelsin a plant.

B. Decreasing Alkaloids by Suppressing MPO and at Least One of A622,NBB1, QPT, and PMT.

Previous reports indicate that suppressing more than one alkaloidbiosynthesis gene in Nicotiana decreases nicotinic alkaloid contentfurther than suppressing only one. For example, suppressing both A622and NBB1 further reduces nicotine levels than suppressing either A622 orNBB1 WO/2006/109197. Accordingly, the present invention contemplatesfurther decreasing alkaloid content by suppressing MPO and one or moreof A622, NBB1, QPT, and PMT. Pursuant to this aspect of the invention, anucleic acid construct comprising MPO and one or more of A622, NBB1,QPT, and PMT is introduced into a cell or plant. An illustrative nucleicacid construct may comprise both MPO and QPT.

II. Increasing Alkaloid Production in Plants

A. Increasing Alkaloids by Overexpressing MPO.

The present invention also relates to increasing alkaloids in plants byoverexpressing MPO. The MPO gene or its open reading frame may be usedto engineer overproduction of pharmaceutical alkaloids, for examplepyrrolidine alkaloids (e.g. nicotine) and/or tropane alkaloids (e.g.scopolamine and cocaine), in plants.

B. Increasing Alkaloids by Overexpressing MPO and at Least One of PMT,QPT, A622 and NBB1.

WO 2005/018307 discusses methods by which alkaloids, such as nicotine,can be further increased by overexpressing more than one gene in thealkaloid biosynthesis pathway. Sato, F., et al. Metabolic engineering ofplant alkaloid biosynthesis. Proc Natl Acad Sci USA. 98(1):367-72(2001). Therefore, the present invention contemplates thatoverexpressing MPO and at least one additional gene in the alkaloidbiosynthesis pathway, such as PMT, will result in greater alkaloidproduction than up-regulating MPO alone.

Pursuant to this aspect of the invention, a nucleic acid constructcomprising MPO and at least one of A622, NBB1, QPT, and PMT isintroduced into a plant cell. An illustrative nucleic acid construct maycomprise, for example, both MPO and PMT. Similarly, for example, agenetically engineered plant overexpressing MPO and PMT may be producedby crossing a transgenic plant overexpressing MPO with a transgenicplant overexpressing PMT. Following successive rounds of crossing andselection, a genetically engineered plant overexpressing MPO and PMT canbe selected.

III. Producing MPO Enzyme

MPO can be introduced into a host cell, thereby producing MPO enzyme inan organism or cell that does not produce this enzyme otherwise. Avariety of products can be produced from these engineered organisms andcells, including alkaloids, alkaloid precursors, alkaloid analogs, andalkaloid biosynthesis enzymes. These products may include nicotine,nicotine precursors, nicotine analogs, and nicotine biosynthesisenzymes. Since MPO catalyzes a key step in the pathway leading totropane alkaloids (e.g. scopolamine and cocaine), cells containing anintroduced MPO gene can also be used to produce tropane alkaloids,tropane alkaloid precursors, tropane analogs, and tropane biosyntheticenzymes. Illustrative host cells include but are not limited to greenplant cells, bacteria, yeast, filamentous fungi, algae, and mammaliancells.

Alkaloid Biosynthesis Sequences

Alkaloid biosynthesis genes have been identified in several plantspecies, exemplified by Nicotiana plants. Accordingly, the presentinvention embraces any nucleic acid, gene, polynucleotide, DNA, RNA,mRNA, or cDNA molecule that is isolated from the genome of a plantspecies, or produced synthetically, that increases alkaloidbiosynthesis. Additionally, expression of such alkaloid biosynthesissequence produces alkaloids in a cell, such as an insect cell. The DNAor RNA may be double-stranded or single-stranded. Single-stranded DNAmay be the coding strand, also known as the sense strand, or it may bethe non-coding strand, also called the anti-sense strand.

It is understood to one skilled in the art that MPO of the presentinvention includes the sequences set forth in SEQ ID NO: 1 and SEQ IDNO: 2, including fragments thereof at least about 21 consecutivenucleotides, which are of a sufficient length as to be useful inductionof gene silencing in plants (Hamilton, A J and Baulcombe, D C Science286, 950-952 (1999)). The invention includes as well as nucleic acidmolecules comprised of “variants” of SEQ ID NO: 1 and SEO ID NO: 2, withone or more bases deleted, substituted, inserted, or added, whichvariant codes for a polypeptide with alkaloid biosynthesis activity.Accordingly, sequences having “base sequences with one or more basesdeleted, substituted, inserted, or added” retain physiological activityeven when the encoded amino acid sequence has one or more amino acidssubstituted, deleted, inserted, or added. Additionally, multiple formsof MPO may exist, which may be due to post-translational modification ofa gene product, or to multiple forms of the MPO gene. Nucleotidesequences that have such modifications and that code for an alkaloidbiosynthesis enzyme are included within the scope of the presentinvention.

For example, the poly A tail or 5′- or 3′-end, nontranslated regions maybe deleted, and bases may be deleted to the extent that amino acids aredeleted. Bases may also be substituted, as long as no frame shiftresults. Bases also may be “added” to the extent that amino acids areadded. It is essential, however, that any such modification does notresult in the loss of alkaloid biosynthesis enzyme activity. A modifiedDNA in this context can be obtained by modifying the DNA base sequencesof the invention so that amino acids at specific sites are substituted,deleted, inserted, or added by site-specific mutagenesis, for example.Zoller & Smith, Nucleic Acid Res. 10: 6487-500 (1982).

An alkaloid biosynthesis sequence can be synthesized ab initio from theappropriate bases, for example, by using an appropriate protein sequencedisclosed herein as guide to create a DNA molecule that, thoughdifferent from the native DNA sequence, results in the production of aprotein with the same or similar amino acid sequence. This type ofsynthetic DNA molecule is useful when introducing a DNA sequence into anon-plant cell, coding for a heterologous protein, that reflectsdifferent (non-plant) codon usage frequencies and, if used unmodified,can result in inefficient translation by the host cell.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in a DNAconstruct are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells or DNAmolecules that are purified. partially or substantially, in solution.Isolated RNA molecules include in vitro RNA transcripts of the DNAmolecules of the present invention. Isolated nucleic acid molecules,according to the present invention, further include such moleculesproduced synthetically.

A “chimeric nucleic acid” comprises a coding sequence or fragmentthereof linked to a nucleotide sequence that is different from thenucleotide sequence with which it is associated in cells in which thecoding sequence occurs naturally.

“Heterologous nucleic acid” refers to a nucleic acid, DNA or RNA, whichhas

been introduced into a cell (or the cell's ancestor) which is not a copyof a sequence naturally found in the cell into which it is introduced.Such heterologous nucleic acid may comprise segments that are a copy ofa sequence which is naturally found in the cell into which it has beenintroduced, or fragments thereof.

“Endogenous nucleic acid” or “endogenous sequence” is “native” to, i.e.,indigenous to, the plant or organism that is to be geneticallyengineered. It refers to a nucleic acid, gene, polynucleotide, DNA, RNA,mRNA, or cDNA molecule that is present in the genome of a plant ororganism that is to be genetically engineered.

“Exogenous nucleic acid” refers to a nucleic acid, DNA or RNA, which hasbeen introduced into a cell (or the cell's ancestor) through the effortsof humans. Such exogenous nucleic acid may be a copy of a sequence whichis naturally found in the cell into which it was introduced, orfragments thereof.

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer, such as the Model 3730xl from Applied Biosystems, Inc.Therefore, as is known in the art for any DNA sequence determined bythis automated approach, any nucleotide sequence determined herein maycontain some errors. Nucleotide sequences determined by automation aretypically at least about 95% identical, more typically at least about96% to at least about 99.9% identical to thoactual nucleotide sequenceof the sequenced DNA molecule. The actual sequence can be more preciselydetermined by other approaches including manual DNA sequencing methodswell known in the art. As is also known in the art, a single insertionor deletion in a determined nucleotide sequence compared to the actualsequence will cause a frame shift in translation of the nucleotidesequence such that the predicted amino acid sequence encoded by adetermined nucleotide sequence may be completely different from theamino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

For the purpose of the invention, two sequences hybridize when they forma double-stranded complex in a hybridization solution of 6×SSC, 0.5%SDS, 5×Denhardt's solution and 100 μg of non-specific carrier DNA. SeeAusubel et al., supra, at section 2.9, supplement 27 (1994). Sequencesmay hybridize at “moderate stringency,” which is defined as atemperature of 60° C. in a hybridization solution of 6×SSC, 0.5% SDS,5×Denhardt's solution and 100 μg of non-specific carrier DNA. For “highstringency” hybridization, the temperature is increased to 68° C.Following the moderate stringency hybridization reaction, thenucleotides are washed in a solution of 2×SSC plus 0.05% SDS for fivetimes at room temperature, with subsequent washes with 0.1×SSC plus 0.1%SDS at 60° C. for 1 h. For high stringency, the wash temperature isincreased to 68° C. For the purpose of the invention, hybridizednucleotides are those that are detected using 1 ng of a radiolabeledprobe having a specific radioactivity of 10,000 cpm/ng, where thehybridized nucleotides are clearly visible following exposure to X-rayfilm at −70° C. for no more than 72 hours.

“Sequence identity” or “identity” in the context of two polynucleotide(nucleic acid) or polypeptide sequences includes reference to theresidues in the two sequences which are the same when aligned formaximum correspondence over a specified region. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties, such ascharge and hydrophobicity, and therefore do not change the functionalproperties of the molecule. Where sequences differ in conservativesubstitutions, the percent sequence identity may be adjusted upwards tocorrect for the conservative nature of the substitution. Sequences whichdiffer by such conservative substitutions are said to have “sequencesimilarity” or “similarity.” Means for making this adjustment arewell-known to those of skill in the art. Typically this involves scoringa conservative substitution as a partial rather than a full mismatch,thereby increasing the percentage sequence identity. Thus, for example,where an identical amino acid is given a score of 1 and anon-conservative substitution is given a score of zero, a conservativesubstitution is given a score between zero and 1. The scoring ofconservative substitutions is calculated, for example, according to thealgorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4: 11-17(1988), as implemented in the program PC/GENE (Intelligenetics, MountainView, Calif., USA).

Use in this description of a percentage of sequence identity denotes avalue determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The present application is directed to such nucleic acid molecules whichare at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to a nucleic acid sequence described in any of SEQ IDNO: 1-2. Preferred are nucleic acid molecules which are at least 95%,96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence shownin any of SEQ ID NO: 1-2. Differences between two nucleic acid sequencesmay occur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among nucleotides in the reference sequence or inone or more contiguous groups within the reference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a referencenucleotide sequence refers to a comparison made between two moleculesusing standard algorithms well known in the art and can be determinedconventionally using publicly available computer programs such as theBLASTN algorithm. See Altschul et al., Nucleic Acids Res. 25: 3389-402(1997).

The present invention further provides nucleic acid molecules comprisingthe nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2, which encodean active alkaloid biosynthesis enzyme, wherein the enzyme has aminoacid sequence that corresponds to SEQ ID NO: 3, and wherein the proteinof the invention encompasses amino acid substitutions, additions anddeletions that do not alter the function of the alkaloid biosynthesisenzyme.

A “variant” is a nucleotide or amino acid sequence that deviates fromthe standard, or given, nucleotide or amino acid sequence of aparticular gene or protein. The terms “isoform,” “isotype,” and “analog”also refer to “variant” forms of a nucleotide or an amino acid sequence.An amino acid sequence that is altered by the addition, removal, orsubstitution of one or more amino acids, or a change in nucleotidesequence, may be considered a “variant” sequence. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. A variant may have “nonconservative” changes, e.g.,replacement of a glycine with a tryptophan. Analogous minor variationsmay also include amino acid deletions or insertions, or both. Guidancein determining which amino acid residues may be substituted, inserted,or deleted may be found using computer programs well known in the artsuch as Vector NTI Suite (InforMax, MD) software. “Variant” may alsorefer to a “shuffled gene” such as those described in Maxygen-assignedpatents (e.g. U.S. Pat. No. 6,602,986).

Methodology for Reducing Alkaloids

In one aspect of the invention, methods and constructs are provided forreducing MPO activity, reducing alkaloid levels, and producing reducedalkaloid plants. While any method may be used for reducing alkaloidlevels, the present invention contemplates antisense, senseco-suppression, RNAi, artificial microRNA, virus-induced gene silencing(VIGS), antisense, sense co-suppression, and targeted mutagenesisapproaches.

RNAi techniques involve stable transformation using RNAi plasmidconstructs (Helliwell and Waterhouse, Methods Enzymol. 392:24-35(2005)). Such plasmids are composed of a fragment of the target gene tobe silenced in an inverted repeat structure. The inverted repeats areseparated by a spacer, often an intron. The RNAi construct driven by asuitable promoter, for example, the Cauliflower mosaic virus (CaMV) 35Spromoter, is integrated into the plant genome and subsequenttranscription of the transgene leads to an RNA molecule that folds backon itself to form a double-stranded hairpin RNA. This double-strandedRNA structure is recognized by the plant and cut into small RNAs (about21 nucleotides long) called small interfering RNAs (siRNAs). siRNAsassociate with a protein complex (RISC) which goes on to directdegradation of the mRNA for the target gene.

Artificial microRNA (amiRNA) techniques exploit the microRNA (miRNA)pathway that functions to silence endogenous genes in plants and othereukaryotes (Schwab et al., Plant Cell 18:1121-33 (2006); Alvarez et al,Plant Cell 18:1134-51 (2006)). In this method, 21 nucleotide longfragments of the gene to be silenced are introduced into a pre-miRNAgene to form a pre-amiRNA construct. The pre-miRNA construct istransferred into the plant genome using transformation methods apparentto one skilled in the art. After transcription of the pre-amiRNA,processing yields amiRNAs that target genes, which share nucleotideidentity with the 21 nucleotide amiRNA sequence.

In RNAi silencing techniques, two factors can influence the choice oflength of the fragment. The shorter the fragment the less frequentlyeffective silencing will be achieved, but very long hairpins increasethe chance of recombination in bacterial host strains. The effectivenessof silencing also appears to be gene dependent and could reflectaccessibility of target mRNA or the relative abundances of the targetmRNA and the hoRNA in cells in which the gene is active. A fragmentlength of between 100 and 800 bp, preferably between 300 and 600 bp, isgenerally suitable to maximize the efficiency of silencing obtained. Theother consideration is the part of the gene to be targeted. 5′ UTR,coding region, and 3′ UTR fragments can be used with equally goodresults. As the mechanism of silencing depends on sequence homologythere is potential for cross-silencing of related mRNA sequences. Wherethis is not desirable a region with low sequence similarity to othersequences, such as a 5′ or 3′ UTR, should be chosen. The rule foravoiding cross-homology silencing appears to be to use sequences that donot have blocks of sequence identity of over 20 bases between theconstruct and the non-target gene sequences. Many of these sameprinciples apply to selection of target regions for designing amiRNAs.

Virus-induced gene silencing (VIGS) techniques are a variation of RNAitechniques that exploits the endogenous antiviral defenses of plants.Infection of plants with recombinant VIGS viruses containing fragmentsof host DNA leads to post-transcriptional gene silencing for the targetgene. In one embodiment, a tobacco rattle virus (TRV) based VIGS systemcan be used. Tobacco rattle virus based VIGS systems are described forexample, in Baulcombe D. C., Curr, Opin. Plant Biol. 2: 109-113 (1999);Lu R, et al., Methods 30: 296-303 (2003); Ratcliff F. et al., The PlantJournal 25:237-245 (2001); and U.S. Pat. No. 7,229,829

Antisense techniques involve introducing into a plant an antisenseoligonucleotide that will bind to the messenger RNA (mRNA) produced bythe gene of interest. The “antisense” oligonucleotide has a basesequence complementary to the gene's messenger RNA (mRNA), which iscalled the “sense” sequence. Activity of the sense segment of the mRNAis blocked by the anti-sense mRNA segment, thereby effectivelyinactivating gene expression. Application of antisense to gene silencingin plants is described in more detail in Stam et al., Plant J. 21:27-42(2000).

Sense co-suppression techniques involve introducing a highly expressedsense transgene into a plant resulting in reduced expression of both thetransgene and the endogenous gene (Depicker and van Montagu, Curr OpinCell Biol 9:373-82 (1997)). The effect depends on sequence identitybetween transgene and endogenous gene.

Targeted mutagenesis techniques, for example TILLING (Targeting InducedLocal Lesions IN Genomes) and “delete-a-gene” using fast-neutronbombardment, may be used to knockout gene function in a plant (Henikoff,of al., Plant Physiol 135:630-6 (2004); Li et al. Plant J. 27:235-242(2001)). TILLING involves treating seeds or individual cells with amutagen to cause point mutations that are then discovered in genes ofinterest using a sensitive method for single-nucleotide mutationdetection. Detection of desired mutations (e.g. mutations resulting inthe inactivation of the gene product of interest) may be accomplished,for example, by PCR methods. For example, oligonucleotide primersderived from the gene of interest may be prepared and PCR may be used toamplify regions of the gene of interest from plants in the mutagenizedpopulation. Amplified mutant genes may be annealed to wild-type genes tofind mismatches between the mutant genes and wild-type genes. Detecteddifferences may be traced back to the plants which had the mutant genethereby revealing which mutagenized plants will have the desiredexpression (e.g. silencing of the gene of interest). These plants maythen be selectively bred to produce a population having the desiredexpression. TILLING can provide an allelic series that includes missenseand knockout mutations, which exhibit reduced expression of the targetedgene. TILLING is touted as a possible approach to gene knockout thatdoes not involve introduction of transgenes, and therefore may be moreacceptable to consumers. Fast-neutron bombardment induces mutations,i.e. deletions, in plant genomes that can also be detected using PCR ina manner similar to TILLING.

Nucleic Acid Constructs

In accordance with one aspect of the invention, a sequence thatsuppresses alkaloid biosynthesis is incorporated into a nucleic acidconstruct that is suitable for introducing into a plant or cell. Thus,such a nucleic acid construct can be used to suppress MPO, andoptionally at least one of A622, NBB1, PMT, and OPT in a plant or cell.

In another aspect of the invention, a sequence that increases alkaloidbiosynthesis is incorporated into a nucleic acid construct that issuitable for introducing into a plant or cell. Thus, such a nucleic acidconstruct can be used to overexpress MPO, and optionally at least one ofA622, NBB1, PMT, and QPT in a plant, as well as express MPO andoptionally at least one of A622, OPT, PMT, and NBB1, for example, in acell.

Recombinant nucleic acid constructs may be made using standardtechniques. For example, the DNA sequence for transcription may beobtained by treating a vector containing said sequence with restrictionenzymes to cut out the appropriate segment. The DNA sequence fortranscription may also be generated by annealing and ligating syntheticoligonucleotides or by using synthetic oligonucleotides in a polymerasechain reaction (PCR) to give suitable restriction sites at each end. TheDNA sequence then is cloned into a vector containing suitable regulatoryelements, such as upstream promoter and downstream terminator sequences.

An important aspect of the present invention is the use of nucleic acidconstructs wherein an alkaloid biosynthesis-encoding sequence isoperably linked to one or more regulatory sequences, which driveexpression of the alkaloid biosynthesis-encoding sequence in certaincell types, organs, or tissues without unduly affecting normaldevelopment or physiology.

“Promoter” connotes a region of DNA upstream from the start oftranscription that is involved in recognition and binding of RNApolymerase and other proteins to initiate transcription. A “constitutivepromoter” is one that is active throughout the life of the plant andunder most environmental conditions. Tissue-specific, tissue-preferred,cell type-specific, and inducible promoters constitute the class of“non-constitutive promoters,” “Operably linked” refers to a functionallinkage between a promoter and a second sequence, where the promotersequence initiates and mediates transcription of the DNA sequencecorresponding to the second sequence. In general, “operably linked”means that the nucleic acid sequences being linked are contiguous.

Promoters useful for expression of a nucleic acid sequence introducedinto a cell to either decrease or increase expression of MPO, A622,NBB1, PMT, or QPT may be constitutive promoters, such as the carnationetched ring virus (CERV), cauliflower mosaic virus (CaMV) 35S promoter,or more particularly the double enhanced cauliflower mosaic viruspromoter, comprising two CaMV 35S promoters in tandem (referred to as a“Double 35S” promoter). Tissue-specific, tissue-preferred, celltype-specific, and inducible promoters may be desirable under certaincircumstances. For example, a tissue-specific promoter allows foroverexpression in certain tissues without affecting expression in othertissues.

Preferred promoters include promoters which are active in root tissues,such as the tobacco RB7promoter (Hsu et al. Pestic. Sci. 44: 9-19(1995); U.S. Pat. No. 5,459,252), maize promoter CRWAQ81 (US publishedpatent application 20050097633); the Arabidopsis ARSK1 promoter (Hwangand Goodman, Plant J 8:37-43 (1995)), the maize MR7 promoter (U.S. Pat.No. 5,837,848), the maize ZRP2 promoter (U.S. Pat. No. 5,633,363), themaize MTL promoter (U.S. Pat. Nos. 5,466,785 and 6,018,099) the maizeMRS1, MRS2, MRS3, and MRS4 promoters (U.S. Pat. App. 20050010974), anArabidopsis cryptic promoter (U.S. Pat. App. 20030106105) and promotersthat are activated under conditions that result in elevated expressionof enzymes involved in nicotine biosynthesis such as the tobacco RD2promoter (U.S. Pat. No. 5,837,876), PMT promoters (Shoji T. et al.,Plant Cell Physiol. 41: 831-39 (2000b); WO 2002/038588) or an A622promoter (Shoji T. et al., Plant Mol Biol. 50: 427-40 (2002)).

The vectors of the invention may also contain termination sequences,which are positioned downstream of the nucleic acid molecules of theinvention, such that transcription of mRNA is terminated, and polyAsequences added. Exemplary of such terminators include Agrobacteriumtumefaciens nopaline synthase terminator (Tnos), Agrobacteriumtumefaciens mannopine synthase terminator (Tmas) and the CaMV 35Sterminator (T35S). Particularly preferred termination regions for useaccording to the invention include the pea ribulose bisphosphatecarboxylase small subunit termination region (TrbcS) or the Tnostermination region. The expression vector also may contain enhancers,start codons, splicing signal sequences, and targeting sequences.

Expression vectors of the invention may also contain a selection markerby which transformed cells can be identified in culture. The marker maybe associated with the heterologous nucleic acid molecule, i.e., thegene operably linked to a promoter. As used herein, the term “marker”refers to a gene encoding a trait or a phenotype that permits theselection of, or the screening for, a plant or cell containing themarker. In plants, for example, the marker gene will encode antibioticor herbicide resistance. This allows for selection of transformed cellsfrom among cells that are not transformed or transfected.

Examples of suitable selectable markers include adenosine deaminase,dihydrofolate reductase, hygromycin-B-phosphotransferase, thymidinekinase, xanthine-guanine phospho-ribosyltransferase, glyphosate andglufosinate resistance, and amino-glycoside 3′-O-phosphotransferase(kanamycin, neomycin and G418 resistance). These markers may includeresistance to G418, hygromycin, bleomycin, kanamycin, and gentamicin.The construct may also contain the selectable marker gene Bar thatconfers resistance to herbicidal phosphinothricin analogs like ammoniumgluphosinate. Thompson et al., EMBO J. 9: 2519-23 (1987). Other suitableselection markers are known as well.

Visible markers such as green florescent protein (GFP) may be used.Methods for identifying or selecting transformed plants based on thecontrol of cell division have also been described. See WO 2000/052168and WO 2001/059086.

Replication sequences, of bacterial or viral origin, may also beincluded to allow the vector to be cloned in a bacterial or phage host.Preferably, a broad host range prokaryotic origin of replication isused. A selectable marker for bacteria may be included to allowselection of bacterial cells bearing the desired construct. Suitableprokaryotic selectable markers also include resistance to antibioticssuch as kanamycin or tetracycline.

Other nucleic acid sequences encoding additional functions may also bepresent in the vector, as is known in the art. For instance, whenAgrobacterium is the host, T-DNA sequences may be included to facilitatethe subsequent transfer to and incorporation into plant chromosomes.

Such gene constructs may suitably be screened for activity bytransformation into a host plant via Agrobacterium and screening formodified alkaloid levels.

Suitably, the nucleotide sequences for the genes may be extracted fromthe Genbank™ nucleotide database and searched for restriction enzymesthat do not cut. These restriction sites may be added to the genes byconventional methods such as incorporating these sites in PCR primers orby sub-cloning.

Preferably, constructs are comprised within a vector, most suitably anexpression vector adapted for expression in an appropriate host (plant)cell. It will be appreciated that any vector which is capable ofproducing a plant comprising the introduced DNA sequence will besufficient.

Suitable vectors are well known to those skilled in the art and aredescribed in general technical references such as Pouwels et al.,Cloning Vectors. A Laboratory Manual, Elsevier, Amsterdam (1986).Particularly suitable vectors include the Ti plasmid vectors.

Genetic Engineering and Selection

The present invention comprehends the genetic manipulation of a plant orcell for regulating alkaloid synthesis via introducing a polynucleotidesequence that encodes an enzyme in the alkaloid synthesis pathway.Accordingly, the present invention provides methodology and constructsfor reducing or increasing alkaloid synthesis in a plant. Additionally,the invention provides methods for producing alkaloids and relatedcompounds in a host cell, such as bacteria, yeast, filamentous fungi,algae, green plants, and mammalian cells.

“Genetic engineering” encompasses any methodology for introducing anucleic acid or specific mutation into a host organism. For example, aplant is genetically engineered when it is transformed with apolynucleotide sequence that suppresses expression of a gene, such thatexpression of a target gene is reduced compared to a control plant. Aplant is genetically engineered when a polynucleotide sequence isintroduced that results in the expression of a novel gene in the plant,or an increase in the level of a gene product that is naturally found inthe plants. In the present context, “genetically engineered” includestransgenic plants and plant cells, as well as plants and plant cellsproduced by means of targeted mutagenesis effected, for example, throughthe use of chimeric RNA/DNA oligonucleotides, as described by Beetham atal., Proc. Natl. Acad. Sci. USA 96: 8774-8778 (1999) and Zhu et al.,Proc Natl Acad Sci USA. 96:8768-8773 (1999), or so-called“recombinagenic olionucleobases,” as described in PCT application WO2003/013226. Likewise, a genetically engineered plant or plant cell maybe produced by the introduction of a modified virus, which, in turn,causes a genetic modification in the host, with results similar to thoseproduced in a transgenic plant, as described herein. See, e.g., U.S.Pat. No. 4,407,956. Additionally, a genetically engineered plant orplant cell may be the product of any native approach (i.e., involving noforeign nucleotide sequences), implemented by introducing only nucleicacid sequences derived from the host plant species or from a sexuallycompatible plant species. See, e.g., U.S. published patent applicationNo. 2004/0107455.

A. Plants

“Plant” is a term that encompasses whole plants, plant organs (e. g.leaves, stems, roots, etc.), seeds, differentiated or undifferentiatedplant cells, and progeny of the same. Plant material includes withoutlimitation seeds suspension cultures, embryos, meristematic regions,callus tissues, leaves, roots, shoots, stems, fruit, gametophytes,sporophytes, pollen, and microspores. The class of plants which can beused in the present invention is generally as broad as the class ofhigher plants amenable to genetic engineering techniques, including bothmonocotyledonous and dicotyledonous plants, as well as gymnosperms. Apreferred nicotine-producing plant includes Nicotiana, Duboisia,Solanum, Anthocercis, and Salpiglessis genera in the Solanaceae or theEclipta and Zinnia genera in the Compositae.

“Tobacco” or “tobacco plant” refers to any species in the Nicotianagenus that produces nicotinic alkaloids, including but are not limitedto the following: Nicotiana acaulis, Nicotiana acuminate, Nicotianaacuminata var. multzjlora, Nicotiana africana, Nicotiana alata,Nicotiana amplexicaulis, Nicotiana arentsii, Nicotiana attenuata,Nicotiana benavidesii, Nicotiana benthamiana, Nicotiana bigelovii,Nicotiana bonariensis, Nicotiana cavicola, Nicotiana clevelandii,Nicotiana cordifolia, Nicotiana corymbosa, Nicotiana debneyi, Nicotianaexcelsior, Nicotiana forgetiana, Nicotiana fragrans, Nicotiana glauca,Nicotiana glutinosa, Nicotiana goodspeedii, Nicotiana gossei, Nicotianahybrid, Nicotiana ingulba, Nicotiana kawakamii, Nicotiana knightiana,Nicotiana langsdorfi, Nicotiana linearis, Nicotiana longiflora,Nicotiana maritima, Nicotiana megalosiphon, Nicotiana miersii Nicotiananoctiflora, Nicotiana nudicaulis, Nicotiana obtusifolia, Nicotianaoccidentalis, Nicotiana occidentalis subsp. hesperis, Nicotianaotophora, Nicotiana paniculata, Nicotiana pauczjlora, Nicotianapetunioides, Nicotiana plumbaginifolia, Nicotiana quadtivalvis,Nicotiana raimondii, Nicotiana repanda, Nicotiana rosulata, Nicotianarosulata subsp. ingulba, Nicotiana rotundifolia, Nicotiana rustica,Nicotiana setchellii, Nicotiana simulans, Nicotiana solanifolia,Nicotiana spegauinii, Nicotiana stocktonii, Nicotiana suaveolens,Nicotiana sylvestris, Nicotiana tabacum, Nicotiana thyrsiflora,Nicotiana tomentosa, Nicotiana tomentosifomis, Nicotiana trigonophylla,Nicotiana umbratica, Nicotiana undulata, Nicotiana velutina, Nicotianawigandioides, and Nicotianax sanderae.

“Tobacco product(s)” refers to a product comprising material produced bya Nicotiana plant, including for example, nicotine gum and patches forsmoking cessation, cigarette tobacco including expanded (puffed) andreconstituted tobacco, cigar tobacco, pipe tobacco, cigarettes, cigars,and all forms of smokeless tobacco such as chewing tobacco, snuff, snusand lozenges.

The Etythroxylaceae (or coca family) is a family of flowering plantsconsisting of 4 genera and about 240 species. The best-known species byfar is the coca (Erythroxylum coca). It has been previously reportedthat when labeled 4-methylaminobutanal diethyl acetal (an acetalderivative of N-methylpyrrolinium cation) was fed to the leaf ofErythroxylum coca, the label was incorporated into the tropane moiety ofcocaine. Leete, Planta Med. 56: 339-352 (1990). Therefore, it isreasonable to expect that the MPO genes of the present invention areinvolved in the formation of cocaine.

As known in the art, there are a number of ways by which genes and geneconstructs can be introduced into plants, and a combination of planttransformation and tissue culture techniques have been successfullyintegrated into effective strategies for creating transgenic cropplants.

These methods, which can be used in the present invention, have beendescribed elsewhere (Potrykus, Annu. Rev, Plant Physiol. Plant Mol.Biol. 42:205-225 (1991); Vasil, Plant Mol. Biol. 5:925-937 (1994);Walden and Wingender, Trends Biotechnol. 13:324-331 (1995); Songstad atal., Plant Cell, Tissue and Organ Culture 40:1-15 (1995)), and are wellknown to persons skilled in the art. For example, one skilled in the artwill certainly be aware that, in addition to Agrobacterium-mediatedtransformation of Arabidopsis by vacuum infiltration (Bechtold et al.,C.R. Acad. Sci. Ser, III Sci. Vie, 316:1194-1199 (1993)) or woundinoculation (Katavic et al., Mol. Gen. Genet. 245:363-370 (1994)), it isequally possible to transform other plant and crop species, usingAgrobacterium Ti-plasmid-mediated transformation (e.g., hypocotyl(DeBlock at al., Plant Physiol. 91:694-701(1989)) or cotyledonarypetiole (Moloney et al., Plant Cell Rep. 8:238-242 (1989) woundinfection), particle bombardment/biolistic methods (Sanford et al., J.Part. Sci. Technol. 5:27-37 (1987); Nehra. et al., Plant J. 5:285-297(1994); Becker et al., Plant J. 5:299-307 (1994)) or polyethyleneglycol-assisted protoplast transformation (Rhodes et al., Science240:204-207 (1988); Shimamoto et al., Nature 335:274-276 (1989))methods.

Agrobacterium rhizogenes may be used to produce transgenic hairy rootscultures of plants, including tobacco, as described, for example, byGuillon et al., Curr. Opin. Plant Biol. 9:341-6 (2006). “Tobacco hairyroots” refers to tobacco roots that have T-DNA from an Ri plasmid ofAgrobacterium rhizogenes integrated in the genome and grow in culturewithout supplementation of auxin and other phytohormones. Tobacco hairyroots produce nicotinic alkaloids as roots of a whole tobacco plant do.

Additionally, plants may be transformed by Rhizobium, Sinorhizobium orMesorhizobium transformation. (Broothaerts et al., Nature 433:629-633(2005)).

After transformation of the plant cells or plant, those plant cells orplants into which the desired DNA has been incorporated may be selectedby such methods as antibiotic resistance, herbicide resistance toleranceto amino-acid analogues or using phenotypic markers.

Various assays may be used to determine whether the plant cell shows achange in gene expression, for example, Northern blotting orquantitative reverse transcriptase PCR (RT-PCR). Whole transgenic plantsmay be regenerated from the transformed cell by conventional methods.Such transgenic plants may be propagated and self-pollinated to producehomozygous lines. Such plants produce seeds containing the genes for theintroduced trait and can be grown to produce plants that will producethe selected phenotype.

Modified alkaloid content, effected in accordance with the presentinvention, can be combined with other traits of interest, such asdisease resistance, pest resistance, high yield or other traits. Forexample, a stable genetically engineered transformant that contains asuitable transgene that modifies alkaloid content may be employed tointrogress a modified alkaloid content trait into a desirablecommercially acceptable genetic background, thereby obtaining a cultivaror variety that combines a modified alkaloid level with said desirablebackground. For example, a genetically engineered tobacco plant withreduced nicotine may be employed to introgress the reduced nicotinetrait into a tobacco cultivar with disease resistance trait, such asresistance to TMV, blank shank, or blue mold. Alternatively, cells of amodified alkaloid content plant of the present invention may betransformed with nucleic acid constructs conferring other traits ofinterest.

B. Cells

The invention contemplates genetically engineering a cell with a nucleicacid sequence encoding an enzyme involved in the production ofalkaloids. Illustrative cells include but are not limited to plantcells, such as Atropa belladonna, Hyoscyamus niger, Arabidopsisthaliana, as well as insect, mammalian, yeast, fungal, algal, orbacterial cells. Suitable host cells are discussed further in Goeddel,GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press,San Diego, Calif. (1990).

“Insect cell” refers to any insect cell that can be transformed with agene encoding an alkaloid biosynthesis enzyme and is capable ofexpressing in recoverable amounts the enzyme or its products.Illustrative insect cells include Sf9 cells (ATCC CRL 1711).

“Fungal cell” refers to any fungal cell that can be transformed with agene encoding an alkaloid biosynthesis enzyme and is capable ofexpressing in recoverable amounts the enzyme or its products.Illustrative fungal cells include yeast cells such as Saccharomycescerevisiae (Baldari, et al., 1987. EMBO J. 6: 229-234) and Pichiapastoris (e.g. P. pastoris KM714 available from Invitrogen). Cells offilamentous fungi such as Aspergillus and Trichoderma may also be used.Archer, et al., Antonie van Leeuwenhoek 65: 245-250 (2004).

“Bacterial cell” refers to any bacterial cell that can be transformedwith a gene encoding an alkaloid biosynthesis enzyme and is capable ofexpressing in recoverable amounts the enzyme or its products.Illustrative bacterial cells include E. coli, such as E. coli strainM15/rep4, which is available commercially from QIAGEN.

“Mammalian cell” refers to any mammalian cell that can be transformedwith a gene encoding an alkaloid biosynthesis enzyme and is capable ofexpressing in recoverable amounts the enzyme or its products.Illustrative mammalian cells include Chinese hamster ovary cells (CHO)or COS cells. Mammalian cells may also include a fertilized oocyte or anembryonic stem cell into which nicotinic alkaloid biosynthesisenzyme-coding sequences have been introduced. Such host cells can thenbe used to create non-human transgenic animals. Examples of systems forregulated expression of proteins in mammalian cells include Clontech'sTet-Off and Tet-On gene expression systems and similar systems. Gossenand Bujard, Proc. Natl. Acad. Sci. USA 89: 55475551 (1992).

“Algae cell” refers to any algae species that can be transformed with agene encoding an alkaloid biosynthesis enzyme without adverselyaffecting normal algae development or physiology. Illustrative algaecells include Chlamydomonas reinhardtii (Mayfield and Franklin, Vaccine23: 1828-1832 (2005).

Since production of alkaloids in an insect cell could adversely affectinsect growth and development, an inducible expression system maymitigate adverse affects. For example, insect cells may be first grownunder non-inducing conditions to a desired state and then expression ofthe enzyme is induced.

Additionally, cells expressing alkaloid biosynthesis genes may besupplied with precursors to increase substrate availability fornicotinic alkaloid synthesis. Cells may be supplied with analogs ofprecursors which may be incorporated into analogs of naturally occurringnicotinic alkaloids.

Constructs according to the invention may be introduced into any cell,using a suitable technique, such as Agrobacterium-mediatedtransformation for plant cells, particle bombardment, electroporation,and polyethylene glycol fusion, calcium phosphate transfection,DEAE-dextran mediated transfection, or cationic lipid-mediatedtransfection.

Such cells may be genetically engineered with a nucleic acid constructof the present invention without the use of a selectable or visiblemarker and transgenic organisms may be identified by detecting thepresence of the introduced construct. The presence of a protein,polypeptide, or nucleic acid molecule in a particular cell can bemeasured to determine if, for example, a cell has been successfullytransformed or transfected. For example, and as routine in the art, thepresence of the introduced construct can be detected by PCR or othersuitable methods for detecting a specific nucleic acid or polypeptidesequence. Additionally, genetically engineered cells may be identifiedby recognizing differences in the growth rate or a morphological featureof a transformed cell compared to the growth rate or a morphologicalfeature of a non-transformed cell that is cultured under similarconditions. See WO 2004/076625.

IV. Quantifying Alkaloid Content

A. Reduced Alkaloids

Pursuant to one aspect of the invention, genetically engineered plantsand cells are characterized by reduced alkaloid content.

A quantitative reduction in alkaloid levels can be assayed by severalmethods, as for example by quantification based on gas-liquidchromatography, high performance liquid chromatography,radio-immunoassays, and enzyme-linked immunosorbent assays. In thepresent invention, alkaloid levels were measured by HPLC analysisperformed on a Waters 2695 separations module equipped with a WatersX-Terra RP18 5 μm 4.6×150 mm with precolumn at a column temperature of60°. The isocratic elution system consisted of 80% A:20% B where solventA consisted of 50 mM citrate, 10 mM octanesulfonic acid pH 3.0 (adjustedwith triethylamine) containing 5% MeOH and solvent B was methanol over15 min at a flow rate of 1 ml/min. Injection volume was 20 μl. Nicotinewas detected at 261 nm via photodiode array detection.

In describing a plant of the invention, the phrase “decreased alkaloidplant” or “reduced alkaloid plant” encompasses a plant that has adecrease in alkaloid content to a level less than 50%, and preferablyless than 10%, or 1% of the alkaloid content of a control plant of thesame species or variety.

B. Increased Alkaloids

In one aspect of the invention, genetically engineered plants and cellsare characterized by increased alkaloid content. Similarly, geneticallyengineered cells are characterized by increased alkaloid production.

In describing a plant of the invention, the phrase “increased alkaloidplant” encompasses a genetically engineered plant that has an increasein alkaloid content greater than 10%, and preferably greater than 50%,100%, or 200% of the alkaloid content of a control plant of the samespecies or variety.

A successfully genetically engineered cell is characterized by nicotinicalkaloid synthesis. For example, an inventive genetically engineeredcell may produce more nicotine compared to a control cell.

A quantitative increase in nicotinic alkaloid levels can be assayed byseveral methods, as for example by quantification based on gas-liquidchromatography, high performance liquid chromatography,radio-immunoassays, and enzyme-linked immunosorbent assays. In thepresent invention, alkaloid levels were measured by high performanceliquid chromatography with a reversed phase column and a photodiodearray detector as described above.

Products

The MPO gene may be used for production of MPO in host cells.Additionally, products can be made using the activity of recombinantMPO. For example, recombinant MPO may be used for the synthesis, orpartial synthesis, of alkaloids. MPO will act on some analogs ofN-methyl putrescine to produce products that do not occur naturally. SeeBoswell of al., Phytochemistry 52:855-869 (1999); Boswell at al.,Phytodhemistry 52:871-878 (1999). Thus, recombinant MPO should be usefulin the production of alkaloid analogs including nicotine analogs. Tothis end, large-scale or commercial quantities of MPO can be produced bya variety of methods, including extracting recombinant enzyme from agenetically engineered plant, cell, or culture system, including but notlimited to hairy root cultures, insect, bacterial, fungal, plant, algae,mammalian cell culture and in vitro translation systems.

In the following examples, functional genomics was used to elucidate anMPO gene that plays an important role in pyrrolidine alkaloid (e.g.nicotine) and tropane alkaloid (e.g. scopolamine and cocaine)biosynthesis. ESTs were obtained from the model plant Nicotianabenthamiana by sequencing subtractive cDNA libraries enriched for genesexpressed in methyljasmonate-treated N. benthamiana roots. The ESTdataset was analyzed for the presence of DNA sequences that may encodeenzymes of the nicotine biosynthetic pathway. Candidate genes weresilenced using virus-induced gene silencing (VIGS), with silencing genesinvolved in nicotine biosynthesis leading to significant reductions innicotine levels.

The data from the present experiments indicate that the gene isolatedfrom Nicotiana benthamiana encodes N-methylputrescine oxidase (MPO),which functions in nicotine biosynthesis. These results show that thecloned MPO belongs to the amine oxidase superfamily which occurs widelyin prokaryotes and eukaryotes and contains a tightly bound Cu II and6-hydroxydopa quinone (TPQ) moiety derived from tyrosine.

These examples are meant to be illustrative only and are not to be readas limiting the present invention.

Example 1 Construction of Subtractive cDNA Libraries, EST Sequencing andSelection of MPO Candidate Genes

Nicotine biosynthesis occurs in the roots of Nicotiana species (Dawson RF, Science 94: 396-397 (1941)) and is induced by insect damage, woundingand the application of jasmonates (Winz R A et al., Plant Physiol. 125:2189-2202 (2001)). In order to identify genes encoding enzyme nicotinebiosynthesis enzymes, a novel approach was used that combined expressedsequence tag (EST) sequencing of methyljasmonate (MeJa)-induced roots ofNicotiana benthamiana with functional analysis using virus-induced genesilencing (VIGS).

Hydroponic Cultivation of Nicotiana benthamiana

Nicotiana benthamiana (Solanaceae) seedlings were grown hydroponicallyin 0.25× Hoagland's solution supplemented with iron chelate solution andoxygenated using an aquarium bubbler. Roots from three-week old plantswere sampled before (t=0) and at 1, 4, and 7 hours after addition ofMeJa to a final concentration of 11 μM. Total RNA was isolated from 450mg each of untreated leaves, untreated roots, and a combinedMeJa-treated root sample composed of 150 mg roots each from the 1, 4 and7 hour time points using a RNeasy midi kit (Qiagen). We constructedthree separate subtractive cDNA libraries: NBREL2, with mRNA pooled fromMeJa-treated roots as tester and untreated root mRNA as driver; NB LEL3,with mRNA pooled from MeJa-treated roots as tester and leaf mRNA asdriver; and NBREL4, with mRNA pooled from MeJa-treated roots as bothtester and driver.

Construction of Subtracted VIGS-cDNA Library

A PCR-select subtractive cDNA library kit (Clontech) was used for cDNAsynthesis with some modifications. Briefly, about 250 μg of total RNAwas mixed with 300 μl of Oligo (dT)₂₅ Dynabeads (Dynal Biotech) inbinding buffer (20 mM Tris-HCl pH 7.5, 1 M LiCl, 2 mM EDTA). After 10min incubation, the beads was washed three times with washing buffer B(10 mM Tris-HCl pH 7.5, 0.15M LiCl, 1 mM EDTA), followed by washingtwice with first strand buffer. The washed beads containing mRNA wasresuspended in 40 μl of cDNA synthesis cocktail (8 μl 5× first strandbuffer, 4 μl 10 mM dNTPs, 24 μl RNase-free water and 4 μl (8U) AMVreverse transcriptase) and incubated at 42° C. for 1.5 hours. The secondstrand synthesis was completed by addition of 120 μl of second strandsynthesis cocktail (32 μl of 5× second strand buffer, 3.2 μl of 10 mMdNTPs, 8 μl of 20× enzyme cocktail and 77 μl RNase free water) andincubation at 16° C. for 2 hours, followed by addition of 4 μl (12U) T4DNA polymerase and further incubation for 30 min. The reaction wasstopped by addition of 20 μl 0.5 M EDTA. The beads were magneticallyseparated, the supernatant removed and the beads resuspended in 500 μlof wash buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl, 1% SDS and10 μg/ml glycogen) and heated at 75° C. for 15 min. The beads were thenwashed three times with wash buffer (5 mM Tris-HCl pH7.5, 0.5 mM EDTA, 1M NaCl and 200 μg/ml BSA), followed by two more washes with Rsal buffer.The beads were resuspended in 84 μl H2O, 10 μl 10× Rsal buffer, 3 μl (30U) Rsal, and incubated at 37° C. overnight. The free cDNA was isolatedby magnetic separation of the beads and was used for adapter ligation,hybridizations and primary PCR as described in the manufacturer'sprotocol. Secondary PCR was performed using primers5′-CGGGATCCTCGAGCGGCCGCCCGGGCAGGT-3′ (BamH1 site underlined) and5′-CGGAATTCAGCGTGGTCGCGGCCGAGGT-3′ (EcoR1 site underlined). ThePCR-select amplified cDNA fragments (700 ng) were digested with EcoRIand BamHI, followed by ligation into a similarly digested TRV-RNA2vector, pYL156 (Liu Y, et al., Plant J. 30:415-429, 2002). The ligationmixture was electroporated into DH10B E. coli competent cells to giveprimary libraries. These was amplified on agar plates, plasmid DNAisolated and used to transform Agrobacterium tumefaciens C58 viaelectroporation. The ligation efficiency as determined by colony PCR was98%.

EST Sequencing of Subtracted VIGS-cDNA Library and Identification of MPGCandidates

To amplify cDNA inserts for sequencing, PCR was performed using vectorprimers 5′-GTTACTCAAGGAAGCACGATGAG-3′ and 5′-CAGTCGAGAATGTCAATCTCGTAG-3′and randomly selected A. tumefaciens colonies as template. The resultingPCR products were sequenced directly using BigDye terminators and theprimer 5′-GTTACTCAAGGAAGCACGATGAG-3′. 2016 ESTs were sequenced fromNBREL2, and 1920 each from NBLEL3 and NBREL4. After removal of poorquality sequences, and combining of the three datasets, we obtained 3480unique transcripts consisting of 606 contigs and 2874 singletons. Thetotal VIGS-EST dataset was annotated via BLASTX comparison to the NCBInon-redundant database.

It has been shown that N-methylputrescine oxidase (MPO), a key enzyme inthe biosynthesis of nicotine and other alkaloids (FIG. 1), is acopper-containing quinoprotein that oxidases N-methylputrescine to formN-methylaminobutanal (Mizusaki S. et al., Phytochemistry 11: 2757-2762,(1972); Davies H M et al., Phytochemistry 28: 1573-1578 (1989)).N-methylaminobutanal spontaneously cyclizes to yield N-methylpyrroliniumion. Using a keyword search of BLASTX results, we identified seven ESTsthat were annotated as copper amine oxidases. The seven clones formedtwo clusters (CL181contig1, 3 members; CL547contig1, 2 members) andthree singletons (Table 1).

Example 2 Cloning of Full-Length cDNA for N-Methylputrescine OxidaseCandidate Gene

cDNA fragments were used for EST sequencing and VIGS analyses andtherefore it was necessary to obtain the full-length cDNA sequence usingRACE PCR,

RACE PCR of Full-Length cDNA for N-Methylputrescine Oxidase CandidateGene

To obtain the 5′ cDNA end of MPO, 5 μg of total RNA from MeJa-treated N.benthamiana roots was reverse transcribed using a GeneRacer kit(invitrogen) according to manufacturers protocol. 5′ RACE PCR reactionswere performed with a GeneRacer 5′ primer and a gene specific primer(5′-CTTGAGCATCTATGGGTGGC-3′) using PCR program (95° C. 2 min; 35 cycles95° C. 30 sec, 58° C. 30 sec, 66° C. 30 sec, 72° C. 1 min; 72° C. 10min) and Pfu Turbo polymerase (Stratagene). The 3′ RACE reaction wereperformed with a GeneRacer 3′ primer and gene specific primer(5′-AGCAATGCGTGACTGTGATCCG-3′) using the same PCR program. The resultingblunt-end PCR products were cloned into the pCR4Blunt-TOPO vector(Invitrogen) and sequenced from both ends.

The full-length MPO gene was 2738 bp in length encoding an open readingframe (ORF) of 2379 bp. The sequence of this MPO gene is set forth inSEQ ID NO: 1. The sequence of the MPO open reading frame (ORF) is setforth in SEQ ID NO: 2. The predicted amino acid sequence is set forth inSEQ ID NO: 3.

Based on computer analysis of primary amino acid sequence (SEQ ID No:3), the Nicotiana MPO contains several domains that are characteristicof copper-containing amine oxidases. Amino acid residues 96-141 formcopper amine oxidase domain N2, and amino acid residues 221-325 formcopper amine oxidase domain N3: A conserved tyrosine at MPO amino acidposition 514 is post-translationally-modified into the redox factortopaquinone (TPQ). Conserved histidine residues at positions 562, 564and 728 may function in copper-binding. As such, it is apparent that anMPO from a corresponding species would likely be more highly conservedthroughout these regions and would contain similar domains. A highdegree of identity across species in these domains provides targetregions in the Nicotiana MPO gene for nucleotide sequences useful in thepreparation of interfering RNA or other gene silencing constructs thatfunction in other related species of plants.

Example 3 Analysis of Expression of MPO Candidate Genes

Nicotine biosynthesis occurs in the roots of Nicotiana species, asevidenced by the root-specific expression of putrescineN-methyltransferase (PMT) and other enzymes catalyzing reactions in thenicotine pathway (Sinclair S J et al., Functional Plant Biol. 31:721-729, (2004)). In order to determine if the cloned MPO was expressedin roots, and was inducible by MeJa, we used reverse transcription-PCR(RT-PCR) to measure the levels of MPO transcript.

RT-PCR Analysis of MPO and PMT Expression in Different N. benthamianaTissues

Reverse transcription-PCR (RT-PCR) was used to determine the expressionof MPO in different N. benthamiana tissues in comparison to PMT, a knownenzyme of the nicotine biosynthetic pathway and a step postulated to beimmediately upstream of MPO (FIG. 1). RNA was isolated from N.benthamiana tissues (young leaf, old leaf, stem, main root, lateral(side) root and whole seedlings) using an RNeasy Plant Mini kit (Qiagen)using the manufacturer's protocol. First strand cDNA was synthesizedfrom 1.7 μg total RNA as described procedure in SuperScript IIIfirst-strand synthesis system for RT-PCR (Invitrogen), followed by PCRamplification of 2 μl cDNA in 50 μl reaction volume using PCR program(95° C. 30 sec, 58° C. 30 sec, 72° C. 50 sec for 30 cycles) and Taqpolymerase. Primers for actin were

5′-CTACAATGAGCTTCGTGTTGC-3′ and 5′-TGCTGAGGGAAGCCAAGATA-3′, for PMT5′-TCATGCTCTTTGAGTCAGCAA′-3′ and 5′-CACCAGTGTTCATTGTTCACT-3′ and for MPO5′-AGGTGGACATCACAGAGGAA-3′ and 5′-AGTCGTTTCAACTCCTCCCGTA-3′.Aliquots of each reaction were analyzed on a 1% agarose gel containingethidium bromide.

As shown in FIG. 2, MPO showed its greatest expression in the fine,lateral roots of N. benthamiana plants. MPO transcript was also detectedin major root tissue and in whole seedlings, which include root and leaftissue. The root-specific expression of MPO therefore supported its rolein nicotine biosynthesis.

qRT-PCR Analysis of MPO and PMT Expression in MeJa-Treated N.benthamiana Roots

The inducibility of MPO by MeJa application was determined usingquantitative RT-PCR (qRT-PCR). The same PCR primers were used as forRT-PCR analysis of expression in different tissues. Total RNA wasextracted from hydroponically grown roots immediately before and 1, 4,and 7 hours after addition of MeJa (11 μM) to the hydroponic solution.Hydroponic conditions are detailed in Example 1. RNA was isolated usinga Plant RNeasy kit (Qiagen). qRT-PCR was carried out as described inSuperScript III platinum SYBR green two-step qRT-PCR kit (Invitrogen)using an iCycler iQ Real-Time detection system (BioRad). Briefly, 1.7 μgtotal RNA was used in the first strand cDNA synthesis in 20 μl reactionvolume, followed by real-time PCR amplification of 1 μl cDNA in 25 μlreaction volume under 95° C. 30 sec, 58° C. 15 sec, 72° C. 50 sec for 40cycles in 96-well optical PCR plate (BioRad). The change of target geneexpression level was obtained using the method of Ramakers et al.(Neuroscience Letters 339: 62-66 (2003)) with actin as a reference genefor transcript normalization.

The change in expression of PMT and MPO in response to MeJa applicationis shown in FIG. 3. MeJa application increased MPO expression 23-foldover pre-induction levels. MPO expression paralleled that of PMT,although the latter showed an even more dramatic increase in transcriptlevels in response to the inducer.

Example 4 Silencing MPO Expression in Plants Using Virus-Induced GeneSilencing (VIGS)

Virus-induced gene silencing (VIGS) was used to test the effect ofsilencing the candidate MPO gene on nicotine biosynthesis. VIGS is afunctional genomics tool that allows for rapid loss- orreduction-of-function experiments in plants (Baulcombe D. C., Curr.Opin. Plant Biol. 2: 109-113 (1999)). The advantages and disadvantagesof VIGS have been reviewed (Lu R, et al., Methods 30: 296-303 (2003)).

VIGS Silencing Constructs Containing MPO Fragments

Three independent VIGS constructs representing different regions of MPOwere tested for their ability to reduce nicotine levels. The positionsof these fragments relative to the MPO ORF are shown in FIG. 4. VIGSconstruct 214D11 was 378 bp in length and corresponded to nucleotidepositions 754-1132 of the full-length MPO cDNA. 214D11 was in thereverse (antisense orientation) relative to the tobacco rattle virus(TRV) coat protein. VIGS construct 317A08 was 252 bp in length andcorresponded to nucleotide positions 1521-1772 of the full-length MPOcDNA. 317A08 was also in the reverse orientation. VIGS construct 403B01was 277 bp in length and corresponded to nucleotide positions 1129-1405of the full-length MPO cDNA. 403B01 was in the forward (sense)orientation relative to the TRV coat protein.

VIGS Methods

N. benthamiana plants were grown in soil in a controlled environmentchamber with 16 hour/23° days and 8 hour/20° nights under approximately100 μmol/m²/s light intensity. Cultures of A. tumefaciens C58 containingthe TRV-RNA1 plasmid or TRV-RNA2 constructs (pYL156) (Liu et at, 2002)were grown overnight at 28° C. After centrifugation, the bacterial cellpellet was resuspended in infiltration buffer containing 1 mM MES (pH5), 10 mM MgCl₂ and 100 μM acetosyringone to OD₆₀₀=1 and allowed tostand at room temperature for 3-6 hours before infiltration. Suspensionsof TRV-RNA1 and TRV-RNA2 constructs were mixed 1:1 and infiltrated intothe underside of the upper leaves of 3-4 week old plants using a 1 mlsyringe. Negative control plants were infiltrated with buffer only or aTRV-RNA2 construct containing a non-functional fragment of greenfluorescent protein (GFP). Plants were grown for 3 weeks before leafnicotine levels were measured using ion-pair HPLC.

Nicotine Analysis by Ion-Pair HPLC

Samples of young (˜3-5 cm) leaves were utilized by excising one half ofa leaf from each plant. After determining the fresh weight of thesample, 200 μl of zirconium beads and 300 μl of 50 mM citrate buffer pH3:methanol (70:30) were added, the sample was homogenized with aBeadbeater followed by incubation in an ultrasonic bath for 10 min. Theresulting extract was incubated at 4° overnight before centrifugationand filtration (0.45 μm, Spin-X) to clarify the extract. Ion-pair HPLCanalysis was performed on a Waters 2695 separations module equipped witha Waters X-Terra RP18 5 μm 4.6×150 mm with precolumn at a columntemperature of 60°. The isocratic elution system consisted of 80% A:20%B where solvent A consisted of 50 mM citrate, 10 mM octanesulfonic acidpH 3.0 (adjusted with triethylamine) containing 5% MeOH and solvent Bwas methanol over 15 min at a flow rate of 1 ml/min. Injection volumewas 20 μl. Nicotine was detected at 261 nm via photodiode arraydetection. Quantification was performed using peak area by comparison toa standard curve (r² 0.999) derived from injection of solutions ofauthentic nicotine ranging in concentration from 1040 μg/ml to 10.4μg/ml.

All three MPO VIGS constructs reduced constitutive nicotine levels ininfected plants (FIG. 5). TRV-GFP control plants had similar nicotinelevels to buffer only treated plants, indicating that TRV infection hadlittle influence on nicotine biosynthesis. We chose the VIGS construct403601 for retesting. Nicotine levels in plants infected with 403601were determined before and five-days after spraying the leaves with MeJa(0.1% (v/v) in water containing 0.1% (v/v) Tween-20) (FIG. 4). Usingconstruct 403601, MeJa-induced nicotine levels were reduced by 77% inMPO silenced plants compared with TRV-GFP controls.

Measurement of N-Methylputrescine Levels in MPO Silenced Plants

Polyamines in the roots of MPO-silenced plants were measured using amethod of Minocha S C et al., (J. Chromatography 511:177-183 (1990)).Briefly, root tissue was frozen in liquid nitrogen and ground to a finepowder. After determining fresh weight of the sample, 200 μl ofzirconium beads and 300 μl of ice cold 5% perchloric acid containing 100nmol/ml of 1,7 diaminoheptane (DAH) were added and the samplehomogenized with a Beadbeater. The extract was clarified bycentrifugation and filtration (0.45 μm, Spin-X). One hundred μl ofsaturated sodium carbonate was added to a 50 μl aliquot of each sample,followed by the addition of 100 μl dansyl chloride (10 mg/ml). Thesamples were mixed by vortexing and then incubated at 60° for 1 hour inthe dark. Fifty μl of proline (100 mg/ml in 5% perchloric acid) wasadded to react with the remaining dansyl chloride. The reaction wasextracted with 400 μl toluene, vortexed and the organic phase separatedby centrifugation. A 200-μl aliquot of the toluene layer was dried bySpeedVac and the residue resuspended in 1 ml of acetonitrile. Ion-pairHPLC analysis was performed on a Waters 2695 separations module equippedwith a Waters X-Terra RP18 5 μm 4.6×150 mm with precolumn at a columntemperature of 40°. The elution solvents consisted of with acetonitrile(solvent A) and 10 mM octanesulfonic acid pH 3.0 (with phosphoric acid)containing 10% acetonitrile (solvent B). A gradient of 30% A to 100% Aover 30 minutes, followed by 3 minutes at 100% A, was used. Injectionvolume was 10 μl and flow rate was 1 ml/min. Detection was performedwith a Waters 2475 fluorescence detector using 340 nm for excitation and510 nm for emission.

N-methylputrescine accumulates in the roots of N. benthamiana plantsinfected with the TRV-MPO silencing construct 214D11 (FIG. 6). Theaccumulation of the substrate for MPO in such plants further supportsour assertion that our VIGS approach has identified the gene for an MPOenzyme that functions in nicotine biosynthesis. N-methylputrescine isalso detectable at lower concentrations in TRV-GFP control plants.

Measurement of MPO Expression in Plants Infected with MPO-VIGSConstructs

qRT-PCR was used to measure the expression of MPO in the roots of plantsthat had been infected with TRV-MPO construct 214D11 or TRV-PMTconstruct. Roots were also sampled from buffer and TRV-GFP controlplants. RNA isolation, cDNA synthesis and qRT-PCR conditions, includingprimer sequences, are described above. MPO and PMT expression increasesin TRV-GFP plants compared to buffer control plants (FIG. 7) while thelevels of MPO transcript are strongly reduced by silencing with TRV-MPO.

Assay of MPO Activity in Plants Infected with MPO-VIGS Constructs

Protein extracts was prepared from the roots of buffer control, TRV-GFPand TRV-MPO (214D11) infected plants. The extraction method described inHashimoto T. et al. (Plant Physiol. 93: 216-221 (1990)) was used.Briefly, 1 g roots was ground in liquid nitrogen and suspended in 20 mlof extraction buffer containing 100 mM potassium phosphate pH 7.5, 0.25M sucrose, 5 mM EDTA, 0.3% ascorbate and 10% PVPP. After centrifugationat 500 g for 30 min, the supernatant was further centrifuged at 11,000 gfor another 30 minutes. The supernatant was subjected to two rounds of20% and 40% ammonium sulphate precipitation. The precipitate wasresuspended in 1 ml water, followed by dialysis overnight in 2 l ofbuffer containing 20 mM potassium phosphate pH 7.5, 1 mM DTT and 20%glycerol. The protein concentration was determined by DC protein assaykit (BioRad). An Amplex red hydrogen peroxide/peroxidase assay kit(Molecular Probes) was used to detect H₂O₂ generated by MPO afteroxidation of N-methyiputrescine. Briefly, a 50 μl reaction containing 8μg of crude protein extract, 1 mM N-methylputrescine and 20 mM potassiumphosphate buffer pH 7.5 was mixed with 50 μl H₂O₂ detection solutioncontaining 0.1 mM Amplex red and 0.2 U/ml peroxidase. After incubatingthe mixture at 30° C. for 30 min, the fluorescence was measured with afluorescence microplate reader (Victor3 UV multilabel reader,PerkinElmer) using excitation at 530-560 nm and emission at 590 nm.

As shown in FIG. 8, MPO activity in the roots of TRV-MPO infected plantswas lower than the activity found in buffer control and TRV-GFP plants.

Example 5 Characterization of the Catalytic Activity of Recombinant MPO

To conclusively show that the cloned gene encoded an MPO enzyme, itsbiochemical properties were characterized and the structure of theproducts formed during incubation of MPO with N-methylputrescine weredetermined.

Expression and Purification of Recombinant MPO

The ORF of the N. benthamiana MPO was expressed in E. coli and therecombinant enzyme was purified for biochemical characterization.Primers 5′-ATGGCCACTACTAAACAGAAAG-3′ and5′-TAGTTTAGCGGCCGCTCAAAGCTTGGCCAGCAAGCT-3′ were designed to amplify theMPO ORF. Using first-strand cDNA as template, the cDNA clone wasamplified using Pfu (Stratagene) and PCR conditions of 95° C. 30 sec,58° C. 30 sec 72° C. 2.5 min for 35 cycles; the product was incubatedwith Taq polymerase 72° C. for 15 min to add A overhangs. The resultingPCR product was cloned into pCR8/GWiTOPO vector (Invitrogen) to generatea Gateway entry clone, which was recombined with destination vectorpHIS8GW by Gateway LR clonase (Invitrogen) to generate the expressionclone pHIS8GW-MPO. The E. coli strain Rosetta (DE3) pLysS (Novagen) wastransformed with pHIS8GW-MPO, which contains an N-terminal octahistidinefusion tag. A single colony was inoculated into 100 ml of overnightexpression autoinduction system (Novagen) containing 50 μg/ml kanamycinand 34 μg/ml of chloramphenicol, and incubated at 28° C. with shakingovernight. Talon Superflow metal affinity resin (Clontech) was used forpurification of recombinant MPO. After centrifugation of the overnightculture, the pellet was resuspended in 10 ml of lysis buffer containing50 mM sodium phosphate buffer pH 8.0, 150 mM NaCl, 0.1% Triton-X100, 5mM imidazole and protease inhibitor cocktail (Novagen). The cellsuspension was sonicated for 2 minutes to lyse the bacteria. Aftercentrifugation of the lysate at 12,000 rpm for 15 minutes, thesupematant was applied onto 200 μl Talon resin column. The column waswashed with 30 ml of wash buffer containing 50 mM sodium phosphate pH7.0, 150 mM NaCl and 10 mM imidazole, followed by elution with 10 ml ofelution buffer containing 50 mM sodium phosphate pH 7.0, 150 mM NaCl and200 mM imidazole. The fractions containing MPO were pooled and dialyzedagainst 2 L of storage buffer containing 50 mM potassium phosphate pH7.5 and 50% glycerol. The protein concentration was determined with DCprotein assay kit (BioRad) and MPO purity was analyzed with SDS-PAGEelectrophoresis (BioRad). One mg of soluble MPO was obtained from 100 mlovernight culture.

Enzyme Assay of Recombinant MPO: Substrate Preferences

Recombinant MPO was assayed using the Amplex red system described above,except that 1 μg of Talon purified MPO was used. To measure the kineticparameters of MPO an assay with two fold serial dilutions ofN-methylputrescine from 10 mM to 0.01 mM at 30° for 30 minutes wasperformed.

Substrate specificity experiments were carried out as above with 1 mM ofN-methylputrescine, putrescine, diaminopropane, cadaverine, spermine andspermidine.

The Km value for N-methylputrescine was determined to be 100 μM.Recombinant MPO preferred N-methylputrescine as a substrate (FIG. 9).The pH optimum of MPO, measured using 0.5 mM N-methylputrescine in 20 mMpotassium phosphate buffer over the pH range 6.5, 7.0, 7.5, 8.0, 8.5 and9.5, was 7.5.

Positive-Ion Electrospray Ionization Mass Spectrometric Analysis(ESI-MS) of MPO Reaction Product

Mass spectrometry (MS) and gas chromatography-mass spectrometry (GC-MS)were used to determine the catalytic product formed by oxidation ofN-methylputrescine by recombinant MPO.

For mass spectrometric analysis, a 50 μl reaction containing 1 μgpurified MPO, 2 mM N-methylputrescine and 20 mM potassium phosphatebuffer pH 7.5 was incubated at 30° C. for one hour, followed by MSanalysis of product composition. A control reaction was performed usingprotein storage buffer instead of MPO solution. MS analysis wasaccomplished with positive-ion electrospray ionization mass spectrometry(ESI-MS) using a tandem quadrupole mass spectrometer (Quattro LC,Micromass, UK) fitted with a pneumatically-assisted electrospray ionsource (Z-spray, Micromass). Samples were introduced by flow injectionusing a binary solvent pump and autosampler (1100 series, HewlettPackard) operating at a flow rate of 20 μL/min. The carrier solventconsisted of 50:50 v/v methanol/water containing 0.1% formic acid.

The reaction product gave a peak of m/z 84 Da, which corresponds to theexpected mass of N-methylpyrrolinium ion (FIG. 10).

GC-MS Analysis (ESI-MS) of MPO Reaction Product

Cyanide trapping of N-methylpyrrolinium cation and GC-MS analysis wascarried out as described in Hashimoto et al., (Plant Physiol. 93:216-221 (1990)). Reaction of N-methylpyrrolinium ion with KCN yields1-methyl-2-cyanopyrrolidine, which can be separated and analyzed byGC-MS. Briefly, the MPO reaction mixture was mixed with 10 μl of 10% KCNsolution and incubated at room temperature for 30 minute, followed byaddition of 100 μl of chloroform. After vortexing, the chloroform phasewas analyzed by GC-MS. Authentic N-methylpyrrolinium cation was treatedwith KCN to yield a 1-methyl-2-cyanopyrrolidine reference compound.GC-MS analysis was accomplished using an Agilent 5973 mass selectivedetector coupled to an Agilent 6890N gas chromatograph equipped with a30-m×0.25-mm DB5MS column with 0.25 μm film thickness (J&W Scientific).The system was controlled by G1701DA MSD ChemStation software. Thechromatography conditions included a split injection (20:1) onto thecolumn using a helium flow of 0.4 ml/mm, an initial temperature of 70°C. for 1 minute, and a subsequent temperature ramp of 10° C./minute to300° C. The mass selective detector was run under standard electronimpact conditions (70 eV), scanning an effective m/z range of 40 to 700at 2.26 scan/s.

GC-MS analysis of the 1-methyl-2-cyanopyrrolidine reference compoundproduced by cyanide trapping of N-methylpyrrolinium ion gave a peak at6.1 min with a molecular ion of m/z 109. A peak of identical retentiontime (6.1 min) and mass spectrum was also present in the MPO reactionmixture. The mass spectra of each peak also had identical diagnosticions that corresponded to those reported for 1-methyl-2-cyanopyrrolidineby Hashimoto et al., Plant Physiol. 93: 216-221 (1990). The detection ofthis product confirms that recombinant MPO catalyzes the oxidation ofN-methylputrescine to form N-methylpyrrolinium ion.

Example 6 Stable Transformation of Plant with an MPO OverexpressionConstruct

Nicotiana benthamiana was transformed with an MPO overexpressionconstruct. Primers 5′-ATGGCCACTACTAAACAGAAAG-3′ and5′-TAGTTTAGCGGCCGCTCAAAGCTTGGCCAGCAAGCT-3′ were designed to amplify theMPO ORF. Using first-strand cDNA as template, the cDNA clone wasamplified using Pfu (Stratagene) and PCR conditions of 95° C. 30 sec,58° C. 30 sec 72° C. 2.5 min for 35 cycles; the product was incubatedwith Taq polymerase 72° C. for 15 min to add A overhangs. The resultingPCR product was cloned into pCR8/GW/TOPO vector (Invitrogen) to generatea Gateway entry clone which was recombined with destination vectorpK7GWG2 by Gateway LR clonase (Invitrogen) to generate the expressionclone pK7GWG2-MPO. The clone was electroporated into Agrobacteriumtumefaciens (C58) and plants were transformed using the leaf disc method(Draper et al, 1988). Transgenic plants were regenerated on selectiveagar media containing kanamycin and the transgenic status was confirmedusing PCR analysis with primers designed to amplify the MPO transgeneand promoter fusion (5′-ACTCCTCCCGTAAAATTTGTGA-3′ and5′-GCGGCCGCACTAGTGATATC-3′) (FIG. 12). Ti seeds were grown in soil andleaf nicotine levels measured using ion-pair HPLC as described above.Nicotine was measured in samples containing three leaf discs (˜50 mgFW), rather than on a fresh weight basis (FIG. 13).

The transgenic plants containing the MPO overexpression construct arescreened to identify plants containing higher amounts of the MPOtranscript and enzyme, and hence MPO activity. The amount of MPOtranscript in transformed plants is determined using Northern blotting,RT-PCR or Real-time qRT-PCR. The amount of MPO enzyme is measured usingWestern blotting with an antibody specifically targeting the MPOprotein, or using a variety of methods for quantifying protein contentincluding proteomic analysis. The amount of MPO enzymatic activity inthe MPO overexpressing plants, as measured by biochemical assay withN-methylputrescine as a substrate, is also used as a way to determine ifthe plants containing the MPO transgene produce higher amounts of theMPO protein. Plants with greater amount of MPO transcript, protein, andenzymatic activity, in comparison to wild-type plants or control plantstransformed with vetor-only constructs, are useful for increasedalkaloid varieties.

1-33. (canceled)
 34. A reduced alkaloid plant produced by a methodselected from: (i) genetically engineering down-regulation ofN-methylputrescine oxidase (MPO) expression relative to a control plant,or (ii) detecting a target mutated plant within a population of mutatedplants, wherein said target mutated plant has a mutation within an MPOgene and decreased expression of or decreased activity of MPO enzymecompared to a control plant.
 35. A reduced alkaloid product producedfrom the plant of claim
 34. 36. The reduced alkaloid plant of claim 34,wherein said alkaloid is a pyrrolidine alkaloid.
 37. The reducedalkaloid plant of claim 34, wherein the reduced alkaloid is nicotine.38. The reduced alkaloid product of claim 35, wherein the reducedalkaloid is nicotine.
 39. The reduced alkaloid product of claim 38,wherein said product is selected from the group consisting of smokingcessation products, cigarettes, cigarette tobacco, cigars, cigartobacco, pipe tobacco, chewing tobacco, snus, tobacco lozenges, foodproducts, food ingredients, feed products, feed ingredients, nutritionalsupplements, and biofuels.